The long-term goal of this proposal is to provide a more comprehensive understanding of signaling pathways and small molecules that impinge them. Mapping information flow in cells is critical to understanding cellular regulation in homeostasis, dysregulation in disease, and the impact of drugs in cells. Transient protein-protein interactions and post-translational modifications (PTMs) are key components of the information flow. However, identification of these interacting partners and especially those for post-translational modifying enzymes remains challenging due to their ephemeral nature and the vast numbers of PTMs in the cell. Current methods employing proteomics and affinity pull-downs are powerful tools for probing protein-protein interactions and PTMs, but these approaches have significant limitations. This proposal aims to address these challenges by engineering and optimizing a new catalytic tagging device, the NEDDylator, which tags its substrates with a stable, simple, and orthogonal mark allowing robust and quantitative identification by proteomics. Our hypothesis is that the NEDDylator technology will be generalizable to exemplary ubiquitin ligases, phosphatases, kinases, and small molecules that affect them, all of which are involved in regulated cell death. The approaches are three-fold: Specific Aim 1: Quantitative and mechanistic analysis of the NEDDylator. The rate-limiting steps and limitations of affinity and product inhibition will be determined for NEDDylation in three complexes: a natural E3-substrate pair, the well-characterized human growth hormone receptor protein complex, and the complex between the drug dasatinib and its target ABL. Specific Aim 2: Engineer the NEDDylator for use in living cells. A fully orthogonal and small molecule inducible NEDDylator will be designed for cellular studies and the proteomic workflow will also be simplified. Specific Aim 3: Elaborate important E3 signaling pathways using the NEDDylator in native proteomes. The NEDDylator will be applied to several pathways of important biological interest in cell death and disease. Information flow will be traced step-by-step through a pathway starting at ubiquitin ligases important for apoptosis and necrosis, and cereblon, a ubiquitin E3 ligase target of the multiple myeloma drug, thalidomide. E3 substrates will be identified and validated, and the NEDDylator will be attached to find their respective cellular binding partners. The proposed studies will validate and expand a novel catalytic tagging platform to dramatically augment the discovery of interacting proteins in extracts and cells. Compared to existing methods, this new technology covalently tags proteins in situ and will enable the discovery of transient as well as high-affinity interactions. The knowledge gained from these studies, both technically and biologically, will likely have a significant impact on our understanding of molecular interactions between proteins and their binding partners in cells.